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A green method

aUniversity of Salento, Department of Eng

73100, Lecce, Italy. E-mail: ermelinda.bloisebInstitute for Bioengineering of Catalonia

Barcelona, Spain. E-mail: lrizzello@ibecbar

† These authors equally contributed to th

Cite this:Nanoscale Adv., 2019, 1, 1193

Received 9th November 2018Accepted 21st December 2018

DOI: 10.1039/c8na00336j

rsc.li/nanoscale-advances

This journal is © The Royal Society of C

for the production of an efficientbioimaging nanotool

Ermelinda Bloise, †*a Maria Pia Di Bello,†a Giuseppe Melea and Loris Rizzello*b

The possibility of exploring basic biological phenomena requires the development of new and efficient bio-

imaging tools. These should ideally combine the feasibility of production (potentially through the use of

green chemistry) together with high targeting efficiency, low cytotoxicity, and optimal contrast

characteristics. In this work, we developed nanovesicles based on cardanol, a natural and renewable

byproduct of the cashew industry, and a fluorescent reporter was encapsulated in them through an

environment-friendly synthesis method. In vitro investigations demonstrated that the cardanol

nanovesicles are efficiently taken-up by both professional and non-professional phagocytic cells, which

have been modeled in our approach by macrophages and HeLa cells, respectively. Co-localization

studies show high affinity of the nanovesicles towards the cell plasma membrane. Moreover, metabolic

assays confirmed that these nanostructures are biocompatible in a specific concentration range, and do

not promote inflammation response in human macrophages. Stability studies carried out at different

temperatures showed that the nanovesicles are stable at both 37 �C and 20 �C, while the formation of

aggregates occurs when the nanodispersion is incubated at 4 �C. The results demonstrate the high

potential of fluorescent cardanol nanovesicles as a green bioimaging tool, especially for investigating cell

membrane dynamics.

Introduction

In the past few decades, nanotechnology has representeda unique opportunity to explore new tools for biomedical appli-cations, especially in bio-imaging and nanomedicine-basedtherapies.1–5 The advancements in materials science havecontributed indeed to developing novel drug delivery systems,some of them being approved for clinical use. Examples include –but are not restricted to – polymer nanoparticles (e.g., PEGylatedproteins), liposome/micellar/protein formulations loaded withdrugs (such as the liposomal amphotericin B lipid complex,micellar estradiol, and albumin-bound paclitaxel nanoparticles),and nanocrystals (e.g., dextran coated SPIONS).6 Different nano-carriers have thus been developed so far to deliver specicpayloads.7 The aim is to improve the drug solubility/stability, andto combine high level of absorption in tissues together witha sustained and controlled release. In this respect, many naturalproducts known to possess anticancer or antimicrobial propertiesneed to be incorporated within nanoparticles to increase theirbioavailability, as well as to reduce potential side effects.8

Degradable natural or synthetic polymers, and more sustainable

ineering for Innovation, Via Arnesano,

@unisalento.it

(IBEC), C/Baldiri Reixac 15-21, 08028

celona.eu; Tel: +34 934 039 956

is work.

hemistry 2019

polymers from renewable resources, have thus been broadlyexploited as preferential startingmaterials for developing deliveryplatforms.9–11 Dextran nanovesicles responsive to acidic pH and/or esterase, and chitosan nanoparticles for cancer targeting areonly some examples.12–14 A recent trend is also the development ofdrug-loaded bioactive nanocarriers made from natural oils (suchas grapeseed oil, sh oil and laurel leaf oil) and surfactantmixtures, as these are able to reduce harmful free radicals anderadicate cancer cells.15

Among the plethora of nature-inspired products that can beexploited as a backbone for delivery systems, cardanol (CA) hasreceived great attention. This is due to the CA structural featuresthat make it suitable for feasible chemical modications. CA isa renewable raw material obtained as a byproduct of the cashewindustry by distillation of the cashew nut shell liquid (CNSL).Several data proved that CNSL and its derivatives possessinteresting in vitro and in vivo biological functions, includingantioxidant, antimicrobial, anti-cancer and larvicidal activi-ties.16–21 CA is able to form a stable nanovesicle dispersion uponcombination with cholesterol,22,23 showing good antioxidantand cytotoxic properties,24 and a potential stabilization capa-bility towards the encapsulated molecules.25 A new challengenowadays is, hence, the possibility of creating a biocompatibledrug delivery system starting from waste materials, which canalso be potentially used as a bioimaging tool.26–30

In this framework, we aimed here to produce CA-basednanovesicles, and to use them as a platform to develop

Nanoscale Adv., 2019, 1, 1193–1199 | 1193

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a green system for imaging biological specimens. We demon-strated that CA-nanovesicles (CANVs) can efficiently encapsu-late rhodamine B (RhB) through an organic solvent-freeprocedure, and be delivered to human macrophages (MF) andHeLa cells, which show an efficient active uptake process.Moreover, we conrmed that these CANVs are biocompatible upto specic concentrations, and do not induce any inammationprocess in MF. Stability studies of the CANVs were performed atthree different temperatures (37, 20 and 4 �C) by monitoring thenanosystem components (CA and CH) over time through HPLC-DAD-MS analyses, coupled with Dynamic Light Scattering(DLS)-based evaluation of size, and z-potential for surfacecharge characterization.

ExperimentalA. Materials

CA was kindly furnished by Oltremare S.r.l. Rhodamine B (RhB)was purchased from Alfa Aesar (Alfa Aesar GmbH&Co, KG,Germany). Cholesterol (CH), potassium chloride (KCl), boricacid (H3BO4), sodium hydroxide (NaOH), acetonitrile (CH3CN),methanol (MeOH) for HPLC, isopropyl alcohol (IPA), and aceticacid were purchased from Sigma-Aldrich®. Ultrapure (UP) waterwas delivered by a Zeneer UP 900 Human Corporation system.Borate buffer solution, pH 9.0, is an UP water solution of H3BO4

30 mM, KCl 70 mM, and NaOH 18 mM. Sonication was carriedout using a Sonorex RK 102H ultrasonic water bath from Ban-delin Electronic. Centrifugation was carried out with a PK121multispeed centrifuge from Thermo Electron Corporation.Cervical human cancer cells (HeLa) and human THP-1 leukemiamonocyte cells were purchased from ATCC®. Dulbecco'sModied Eagle Medium (DMEM), fetal calf serum, L-glutamine,streptomycin, penicillin, trypsin–EDTA solution, RPMI-1640medium, HEPES buffer, thiazolyl blue tetrazolium blue (MTT),phosphate-buffered saline (PBS), 40,6-diamidino-2-phenylindole(DAPI), bacterial lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich®. CellMask was purchased from Life Technologies, andTHP-1 blue NF-kB reporter cells were purchased fromInvivoGen.

B. Preparation of cardanol-based nanovesicles

RhB dye was mixed with CA, CH (molar ratio 0.04 : 1 : 0.6,respectively), and glass beads (10 g, diameter of 4 mm) bymechanical stirring at 200 rpm at 90 �C for 1 h to form a lipidlm on the ask's wall. The resulting lm was hydrated with40 mL of borate buffer at pH 9.0, preheated at 50 �C, undermechanical stirring (800 rpm), and nally heated at 90 �C for1 h. Then, the obtained vesicular nanodispersion was sonicatedfor 15 min at 60 �C, and then centrifuged (7000 rpm for 15 min)to collect the supernatant as the sample. Purication of thenanodispersion from non-entrapped RhB was performed byexhaustive dialysis. The sample was transferred into a dialysistubing cellulose membrane (12 kDa) and then sealed at bothends with clips and dialyzed against borate buffer solution, pH9.0, for some days. Fresh buffer solution was replaced every day,

1194 | Nanoscale Adv., 2019, 1, 1193–1199

and the free RhB was monitored at 554 nm through UV-vismeasurements using a Jasco V-660 spectrophotometer untildisappearance of the signal. A sample without dye was preparedwith the same procedure as that for the blank reference.

C. Dynamic light scattering analysis

Measurements of dynamic light scattering (DLS) and electro-phoretic light scattering were both carried out on a MalvernZetasizer Nano ZS90 on samples diluted with UP water toestablish the size and z-potential of nanovesicles. The hydro-dynamic diameter (d) of the vesicle nanodispersion was deter-mined at 25 �C measuring the autocorrelation function at a 90�

scattering angle. Each d value is the average of three separatemeasurements. The z-potential values of nanovesicles weredetermined at 25 �C from an average of 3 measurements. Thevoltage ramps were performed according to the DLS manufac-turer instructions.

D. HPLC-DAD-MS analysis

Separation and quantication of nanocarrier components (CAand CH) were performed on an Agilent Technologies (Wald-bronn, Germany) modular model 1200 system, consisting ofa vacuum degasser, a binary pump, an autosampler, a xed-temperature column compartment, and a diode array detector(DAD). Natural CA extract is a mixture of compounds and wasused as such as a standard to develop a calibration curve in therange 10–1000 mg L�1. For CA quantication nanovesicles werelysed by adding 5 mL of MeOH to 1 mL of samples, then dried,resuspended in 1 mL of CH3CN, and passed through a 0.22 mmnylon lter before injection. The chromatograms were recordedusing Agilent Mass Hunter soware (rev. B.06.00). Chromatog-raphy separation was carried out on a 150 � 4.6 mm i.d., 5 mmGemini C18 column set at 25 �C. The mobile phase wascomposed of water/acetic acid 0.1% v/v (solvent A) and CH3CN(solvent B). The chromatograms were acquired at a wavelengthof 280 nm working at a ow rate of 0.8 mL min�1, with thefollowing gradient: 0 min, 50% B; 20 min, 100% B; and 32 min,100% B. Three injections were performed for each sample, andthe results are the average of those values.

Qualitative analyses of CA were carried out aer chromato-graphic separation using the same system equipped with a 6540quadrupole time-of-ight (QTOF) mass analyzer with an elec-trospray ionization (ESI) source. The HPLC-ESI-MS analyses ofthe CA components were performed both in negative-ion modeand in positive-ion mode.25 Quantication of CH was performedusing a curve in the range 10–1000 mg L�1. Nanovesicles werelysed by adding 5 mL of MeOH to 1 mL of samples, then dried,resuspended in 1 mL of mixed solvent CH3CN/MeOH/IPA(7 : 3 : 1, v/v/v) and passed through a 0.22 mm nylon lterbefore injection. The chromatograms were recorded using theAgilent Mass Hunter soware mentioned above. Chromatog-raphy separation was carried out on a 150 � 4.6 mm i.d., 5 mmGemini C18 column set at 25 �C. The isocratic mobile phase wascomposed of CH3CN/MeOH/IPA (7 : 3 : 1, v/v/v). The chro-matograms were acquired at a wavelength of 202 nm working at

This journal is © The Royal Society of Chemistry 2019

Fig. 1 Composition of the CA mixture.

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a ow rate of 1 mLmin�1.31 Three injections were performed foreach sample, and the results are the average of those values.

E. Nanovesicle stability studies

Stability studies were carried out on stored CANV samples atthree different temperatures (37 �C, 20 �C and 4 �C) for 3, 4, 7and 10 days. Stability proles were obtained bymonitoring theird and z-potential by DLS analysis and the CA and CH concen-trations by HPLC-DAD. The equation used to calculate thepercentage of CA and CH in nanovesicle samples [analyte (%)] isas follows:

analyte% ¼ Cdays

C0

� 100

where Cdays is the analyte concentration determined at differentdays, whereas C0 is the analyte concentration determined on thefresh sample.

F. Cell uptake and metabolic assay

HeLa cells were cultured and maintained using DMEM con-taining 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 mgmL�1 streptomycin and 100 IU mL�1 penicillin. Cells werecultured at 37 �C/95% air/5% CO2. Cells were periodically sub-cultured using trypsin–EDTA solution 0.25% for the detach-ment process and centrifuged at 300g for 5 min for the pelletcollection. THP-1 cells were cultured and maintained in RPMI-1640 medium, supplemented with 10 mM HEPES buffer, 10%(v/v) fetal calf serum, 2 mM L-glutamine, 100 mg mL�1 strep-tomycin and 100 IU mL�1 penicillin. THP-1 monocytes weredifferentiated to macrophages through incubation with 5 ngmL�1 PMA for 48 hours.32 We chose this PMA concentration asit does not modify the regulation of stress- and inammation-related genes.33 For the metabolic assay, the MTT method wasused. Briey, cells were seeded at a concentration of 5 � 103

cells per well in a 96 well plate overnight. Increasing concen-trations of CANVs were then added in the growthmedia, namely5, 10, 30, 50, and 70 mg mL�1, for 24 and 48 hours. The growthmedium was then removed, and the tetrazolium blue salt wasadded for 2 hours. An acidied solution of isopropanol was thenadded to dissolve the water-insoluble MTT formazan. Thesolubilized blue crystals were measured colorimetrically at570 nm (plate reader ELx800, BioTek). For uptake imaginganalyses, HeLa and THP-1 cells were seeded on glass-bottomPetri dishes (Ibidi) at a concentration of 5 � 103 cells per well,and incubated with RhB-labeled CANVs (0.1 mg mL�1) for 24and 48 hours, followed by 3 steps of PBS washing. Cells werethen stained with DAPI for nuclear staining, and far-red Cell-Mask for cell membrane staining and imaged with a confocalmicroscope (Leica SP8). For the uptake quantication, 10different regions of the Petri dishes were captured, and theuorescent signal was normalized to the nuclear intensitysignal (using ImageJ).

G. Quantication of M(F) inammation levels

For the quantication of MF inammation levels, THP-1 blueNF-kB reporter cells were used. These cells are stably

This journal is © The Royal Society of Chemistry 2019

transfected and express a secreted embryonic alkaline phos-phatase (SEAP) reporter gene driven by an IFN-beta minimalpromoter fused to ve copies of the NF-kB transcription factor,which promotes cytokine production. The translocation of NF-kB from the cytosol to the nucleus induces the release of theSEAP phosphatase, whose activity can be monitored colori-metrically. THP-1 blue NF-kB reporter cells were differentiatedinto MF as previously described, and then incubated with 200ng mL�1 bacterial LPS as a positive control, and with 30 mgmL�1 CANVs for 24 and 48 hours. 20 mL of media were thenremoved and incubated with the incubation media, accordingto the manufacturer’s instructions. The quantication of mediacolor change was performed with a spectrophotometer (platereader ELx800, BioTek) by reading at 570 nm.

Results and discussion

The CA used as the main constituent of the green nanocarrier bya mixture of differently (un)saturated compounds (Fig. 1).Hence, HPLC-DAD-MS analyses were preliminarily used todetermine the CA composition.25 Four main components werefound, corresponding to three unsaturated components, 3-[8(Z),11(Z),14-pentadecatrienyl]phenol (C15:3), 3-[8(Z),11(Z)-pentadecadienyl]phenol (C15:2), and 3-[8(Z)-pentadecadecenyl]phenol (C15:1), and the saturated component 3-n-pentadecyl-phenol (C15:0). For CA quantication, the most abundant mono-unsaturated C15:1 component was considered. The stability ofthe nanovesicles was investigated by monitoring the change ofCA and CH concentrations, and modication in the sizedistribution and z-potential values over time. This was donewhile incubating the CANVs in glass tubes at three differenttemperatures (37 �C, 20 �C and 4 �C). The size distribution (d)and z-potential values for all samples are displayed in Fig. 2A.Nanovesicles are stable at both 37 �C and 20 �C, as their sizedistribution is not affected. However, incubation at 4 �C led toa signicant increase of the mean diameter, thus indicating theformation of aggregates. We have further characterized thechange in the physicochemical parameters of CANVs as a func-tion of temperature, and quantied the variations in CA and CHconcentration over time. These data further indicate thatincubation at 4 �C decreases the stability of CANVs as conrmedby the decrease of CA and CH concentration (Fig. 2B). Trans-mission electron microscopy (TEM)-based analyses conrmed

Nanoscale Adv., 2019, 1, 1193–1199 | 1195

Fig. 2 Physicochemical characterization of CA nanovesicles. (A) Stability studies based on DLS for size distribution (blue graph, quantified on theleft y axis) and z-potential for surface charge analysis (orange graph, quantified on the right y axis). (B) Variations in CA and CH concentration overtime, as a function of environmental temperature.

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that the morphology of the green nanocarriers is characterizedby a very regular spherical structure, as we have already reportedin our previous studies.23,24 Aer analyzing the physicochemicalparameters of the “green” CANVs, and their stability as a func-tion of the environmental temperature, we addressed theirinteractions with cells. As model systems, we used humancervical cancer (HeLa) cells, and leukemic monocytes (THP-1),differentiated to a macrophage (MF) phenotype (see theExperimental section). We encapsulated RhB under solvent-freeconditions within the CANVs. RhB-encapsulated CANVs wereincubated with cells, and the kinetics of uptake were studied byconfocal microscopy (Fig. 3). We have observed an increase inthe relative uorescence intensity in HeLa cells (i.e., the uo-rescence of RhB-encapsulated CANVs normalized by thenumber of cells) from ca. 0.7 to 1, corresponding to 24 and 48hours of incubation, respectively (Fig. 3A). Moreover, thequantication of the Pearson coefficient, which is a measure ofthe spatial overlap between two different signals, conrms thatthere is almost 85% signal co-localization between the nano-vesicles and the plasma membrane. The value rises up to ca.90% aer 48 hours of incubation. This suggests that thenanovesicles tend to interact with the plasma membrane, andwhile some of these will undergo an internalization processthrough endocytosis, a signicant number of nanovesiclesremain at the membrane level. We have also observed similaruptake proles for MF, as the normalized uorescence intensityof the RhB-encapsulated CANVs increased from ca. 0.6 to 1,which corresponds to 24 and 48 hours of incubation with cells(Fig. 3B). In this case, however, the Pearson coefficient had

1196 | Nanoscale Adv., 2019, 1, 1193–1199

lower values with respect to HeLa cells. In particular, it wasabout 62% aer 24 hours and ca. 70% aer 48 hours of incu-bation with nanovesicles (Fig. 3B). This would suggest that thenanovesicles interact less with the plasma membrane of MF

compared to the stronger interaction with the membrane ofHeLa cells. This can be ascribed to the different uptakeprocesses of the two cells. HeLa cells engulf materials only byendocytic processes while MF combine endocytosis withphagocytosis (typical of professional phagocytes). This means,in turn, that the dwelling time of nanoparticles at the plasmamembrane level in MF might be lower compared to that inHeLa because MF are much more efficient in performingnanovesicle engulfment. Aer analyzing the uptake prole ofthe nanovesicles, we addressed their effects on both cells.Hence, we carried out metabolic assays to test the potentialtoxicity of the CANVs as a function of concentration and incu-bation time. CANVs did not induce any decrease in cell viabilityat 5, 10, and 30 mg mL�1 aer 24 hours of incubation with HeLacells (Fig. 4A, le), while the viability dropped to ca. 40% uponincubation with 50 mg mL�1 (hence close to their IC50). Thenanovesicles displayed signicant toxicity at 70 mg mL�1, whereno viable HeLa cells were detected (Fig. 4A, le). Similarly,CANVs did not induce toxicity aer 48 hours at 5 and 10 mgmL�1, while at 30 and 50 mg mL�1 they were slightly more toxicwith respect to the 24 hour case. Also in this case, 70 mg mL�1

was lethal to HeLa. With respect to MF, signicant toxicity wasdetected starting from 50 mg mL�1 CANVs aer 24 and 48 hoursof incubation (Fig. 4A, right). As for HeLa, 70 mg mL�1 CANVsresulted also lethal for MF. We then went deeper into the

This journal is © The Royal Society of Chemistry 2019

Fig. 3 Uptake study of CANVs in HeLa cells (A) and MF (B) after 24 and 48 hours of incubation. The dashed squares are higher magnificationimages of the selected area. Uptake quantification and co-localization studies are reported on the right-hand side.

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potential mechanisms of toxicity and tried to understandwhether nanovesicles induce inammation in MF. To do this,we used THP1-NF-kB reporter cells, which are extremely

This journal is © The Royal Society of Chemistry 2019

powerful to monitor and quantify the level of translocation ofthe transcription factor NF-kB from the cytosol to the nucleus.This translocation occurs when MF have to start an

Nanoscale Adv., 2019, 1, 1193–1199 | 1197

Fig. 4 (A) Metabolic assay (MTT) to test the viability of HeLa cells (left) and MF (right) upon incubation with 5, 10, 30, 50, and 70 mg mL�1 CANVs,after 24 (red) and 48 (blue) hours of incubation. (B) Inflammation measurement: quantification of the SEAP activity in MF, which indicates thelevels of NF-kB translocation from the cytosol to the nucleus.

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inammation process upon external stimuli (like the presenceof bacterial lipopolysaccharides – LPS). The nuclear trans-location of NF-kB induces then the production of cytokines,which are the key players in inammation. The THP1-NF-kBreporter cells used in this work carry a stable integration of anNF-kB-inducible SEAP reporter construct. Hence, we are able toquantify NF-kB translocation/activation through the quanti-cation of the SEAP activity. We thus incubated MF with LPS asa positive control, as it induces a full NF-kB translocation fromthe cytosol to the nucleus, and we normalized the otherconditions according to this treatment (Fig. 4B). We did notdetect any signicant increase in the SEAP activity under all theconditions tested (10, 30, and 50 mg mL�1 CANVs), compared tocontrol untreated cells. This indicates that CANVs do notpromote inammation, which is absolutely crucial for anybiomedical application of these nanosystems.

Conclusions

The current need for exploring and deciphering basic biologicalphenomena requires the development of new and effective bio-imaging nanotools. While doing this, nature-inspired products

1198 | Nanoscale Adv., 2019, 1, 1193–1199

should be used for the design of nano-vehicles as they representa safe, inexpensive, renewable and theoretically unlimitedresource. In this context, we exploited a natural byproduct of thecashew industry, cardanol, and combined it with cholesterol forthe development of cardanol-nanovesicles by a green chemistryapproach. We encapsulated the nano-vesicles with a uorescentdye (rhodamine) and demonstrated that human HeLa and THP-1 (macrophages) cells efficiently uptake them. The nanovesicleshave also been demonstrated to uniformly stain the cell plasmamembrane, a process having high impact in imaging-basedapplications. Finally, we demonstrated also that CANVs arebiocompatible in a specic concentration range, and do notinduce any inammation process in macrophages.

Conflicts of interest

The authors declare that there are no conicts of interest in thiswork.

This journal is © The Royal Society of Chemistry 2019

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Acknowledgements

Ermelinda Bloise thanks Apulian Region Programs for nancialsupport (Intervento conanziato dal Fondo di Sviluppo eCoesione 2007–2013 – APQ Ricerca Regione Puglia “Programmaregionale a sostegno della specializzazione intelligente e dellasostenibilita sociale ed ambientale – Future In Research” –

Grant XYA7HY5). Loris Rizzello acknowledges the MarieSkłodowska-Curie Actions for funding his fellowship andresearch activities.

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